The eukaryotic ATP-ases of SMC family (structural maintenance of chromosomes) form several essential protein complexes that determine the higher-order chromosome structure and dynamics in eukaryotic cells. One of these complexes, termed condensin, is in the current focus of studies by the Unit. Condensin complex constitutes the main molecular machine of chromosome condensation, a process indispensable for proper segregation of sister chromatids during cell division. Condensin is conserved in eukaryotic evolution, and in budding yeast it is composed of five essential subunits: Smc2, Smc4, Ycs5/Ycg1, Ycs4 and Brn1. The vital activity of condensin is to bind DNA and to change its superhelical state, introducing positive DNA supercoiling. Existing in vitro studies do not allow, however, to predict how condensin interacts with a chromatin fiber, i.e. its natural substrate. Thus, the molecular characterization of the authentic binding sites for condensin activity in vivo is an essential step towards elucidating the molecular mechanisms of chromosome condensation. In order to understand the essence of condensin activity in chromatin the studies in the Unit were focused on several particular aspects: (1) the regulation and specificity of condensin targeting to the nucleolar chromatin and (2) the role of active condensin for functionality of other critical chromosomal sites, paricularly centromeres, telomeres and DNA replication terminators.[unreadable] (1) The regulation and specificity of condensin targeting to the nucleolar chromatin. The characterization of the authentic binding sites for condensin activity in vivo is an essential step towards elucidating the molecular mechanisms of chromosome condensation. Furthermore, answering the question how does condensin recognize a specific binding site on a chromosome, requires understanding the molecular roles of post-translational condensin modifications. We previously identified a promising candidate for a condensin regulator - Smt4p, the SUMO isopeptidase (Smt3p in yeast). Our recent data suggest that condensin is regulated by balanced sumoylation, i.e both adding and timely removal of SUMO moiety have specific effects on condensin targeting. To investigate whether condensin itself and/or some other proteins cooperating with condensin are directly modified by Smt3p, we developed an unbiased screening procedure, which enables the identification of the authentic Smt3p conjugates in vivo by utilizing differential-length SUMO tags. Using this SUMO footprint assay we were able to detect SUMO modification of four condensin subunits (Smc2p, Smc4p, Ycs4p and Ycs5p). However, assigning a specific biological role to these modification was challenging, because the finding that condensin subunits are modified, by itself, does not provide insights into the biological function of these modifications. The breakthrough was achieved after we constructed a GFP-Smt3p fusion expressed from the native SMT3 promoter and showed that SUMO conjugates are significantly enriched in the nucleolus the essential hub of activities of condensin and both topoisomerases I and II. Further investigation of the biological role of condensins sumoylation has led to the hypothesis that SUMO modification of condensin facilitates its targeting to the nucleolus, promoting condensin cooperativity with nucleolar pools of sumoylated topoisomerases I and II. We tested this hypothesis with series of experiments: first, we mutated lysine residues to arginine (KR mutations) in all sixteen of the sumoylation consensus sites (spread across four condensin subunits) and assessed whether the generated SUMO-less condensin has any defects in its targeting to the nucleolus, chromosome condensation, chromosome segregation and cell viability. Second, we combined the condensin KR mutants with topoisomerase mutations, which reduced the nucleolar presence of topoisomerases. Third, we identified the SUMO E3 enzyme for condensin, the Mms21 protein, and showed that the significant part of Mms21 E3 activity is to modify condensin and cohesin complexes. As a result of this study, it was established that: 1) SUMO is prominently enriched in the nucleolus; 2) triple SUMO E3 mutants are defective in rDNA segregation and maintenance; 3) genetic interactions between mms21-CH, top1 and top2 mutations signifies a common/redundant pathway in rDNA maintenance; 4) condensin and cohesin binding to rDNA is directly regulated by Mms21-mediated sumoylation; 5) condensin sumoylation is essential in the absence of Top1 and sumoylated Top2. These findings substantiated the paradigm that the SUMO code plays a role in the subnuclear compartmentalization of chromatin proteins and suggested that SUMO-mediated co-targeting of individual proteins to a specific chromosomal compartment can induce their functional cooperation.[unreadable] (2) The role of active condensin for the functionality of critical chromosomal sites: the centromere and kinetochore. ChIP-chip analysis of the genome-wide condensin binding pattern, previously conducted in the Unit, showed that the peri-centromeric regions are enriched in condensin binding. This enrichment suggested that condensins activity might facilitate centromere function. Furthermore, our previous studies confirmed that condensin has some role at centromeres: ChIP-chip analysis uncovered that a notable interphase enrichment of condensin near centromeres is increased in mitosis; the spindle assembly checkpoint (SAC) is the factor responsible for condensin mutant arrest; and both Dsn1 (a part of the MIND complex) and Cse4 (the orthologue of the CENP-A centromeric histone) proteins were partially delocalized from centromeres in condensin mutants. While these analyses have uncovered a putative molecular interface between condensin and centromeres in yeast, it was puzzling why checkpoints did not prevent chromosome nondisjunction and aneuploidy upon condensin dysfunction in humans. Therefore, we generated efficient RNAi knockout of condensin, and analyzed the structure of condensin-defective centromeres and kinetochores biochemically, genetically and cytologically in human cells. Our data showed that the centromere structure was abnormal and that the inter-kinetochore distance was increased (almost two-fold) in SMC2-depleted metaphase chromosomes. Furthermore, upon depletion of condensins I and II in both human and amphibian cells, the resulting cumulative centromeric structural defects indeed activated SAC; however, chromosome missegregation in anaphase was not prevented. While investigating the molecular nature of such a slippage through SAC in human cells we found that depletion of condensin activity results in a significant loss of CENPA from the centromere and a SAC-mediated metaphase delay. As CENP-A loads to centromeres in the previous cell cycle, we analyzed CENP-A enrichment during several cell divisions, using covalent fluorescent pulse-chase labeling (SNAP tagging) in collaboration with D. Cleveland lab. Moreover, the extreme stretching of centromeric chromatin by spindle forces has led to the deformation of both inner kinetochores and the microtubule-capturing module (HEC1 complex), highly resembling merotelic configuration. As a result, Aurora B was mis-localized and partially lost activity in condensin-depleted centromeres, suggesting that the correction of merotelic attachments prior to anaphase onset was severely impaired without condensin. The monastrol recovery experiments showed that the phenotype of the high incidence of merotelic attachment was nearly identical and epistatic in the cases of condensin depletion and Aurora B inactivation, indicating that condensin plays a pivotal role in the proper spatial positioning of the kinetochore relatively to the microtubule-regulating protein complexes in the inner centromere.